This invention relates to a method of screening for precursor lesions which can lead to cervical malignancy, methods of detecting and typing HPV infections, and reagents of use in the above methods.
Papillomaviruses (PVs) cause epithelial tumours in humans which vary in severity depending on the site of infection and the HPV (human papilloma virus) type involved (Laimins-, 1993; Villiers de, 1994). Low risk types such as HPV 1 or HPV63 (Egawa et al. 1993a; Egawa et al. 1993b) cause benign cutaneous warts which progress to malignancy only rarely, while high risk viruses such as HPV 16 and HPV31 cause flat warts at mucosal sites, and are associated with high grade cervical intraepithelial neoplasia (CIN) and cancer (Schneider, 1994). Formation of an HPV-induced tumour is thought to require infection of an epithelial basal cell, and the expression of viral early proteins in order to stimulate cell proliferation. The late stages of the virus life cycle, which ultimately lead to the production of infectious virions, are initiated only as the infected cell migrates through the upper differentiated layers of the epidermis. Viral and cellular events which influence HPV late gene expression have not been characterised as, until recently, there has been no convenient system for mimicking productive infection in vitro (Laimins, 1993).
Studies on naturally-occurring warts have revealed the virus to encode three late proteinsxe2x80x94L1 and L2, which are virion coat proteins (Doorbar et al, 1987), and E1{circumflex over ( )}E4, a non-structural late protein of unknown function (Doorbar et al, 1986). In HPV1-induced warts the E1{circumflex over ( )}E4 protein is first expressed in cells of the lower spinous layer, and assembles into distinctive cytoplasmic and nuclear inclusions. During terminal differentiation it is post-transcriptionally modified by phosphorylationxe2x80x94(Grand et al, 1989) and by removal of sequences from the N-terminus (Doorbar et al, 1988; Roberts et al, 1994). The E1{circumflex over ( )}E4 proteins of high risk viruses have been poorly characterised, because it has been thought that HPV16-induced lesions contain only small numbers of productively infected cells, and that these contain only low levels of E4 (Doorbar et al, 1996b; Crum et al, 1990). A single Mab (TVG 402) to HPV16 E1{circumflex over ( )}E4 has been used to locate the protein to the cytoplasm but was reported not to work well on paraffin-embedded archival material (Doorbar et al, 1992). Furthermore, polyclonal antibody studies on the E4 proteins of mucosal viruses have yielded conflicting results. One study has supported the above findings (Crum et al, 1990), while another has indicated that the protein is located to the nucleus (Palefsky et al. 1991).
In many countries there are screening programmes to detect the presence of cervical carcinoma at an early stage. Generally such programmes operate by obtaining cervical smears from women potentially at risk of developing cervical cancer, with the resulting smears routinely being examined by conventional histopathological techniques. These techniques are laborious and time-consuming, require considerable experience to interpret results correctly, and frequently give rise to relatively large percentages of false positive results, causing unnecessary alarm. False negatives can occur when screening is carried out by inexperienced personnel and can lead to the classification of pre-cancerous lesions as normal. There is thus a need for an improved cervical cancer screening method.
It is well known that there is a very strong correlation between HP-infection and development of cervical carcinoma: over 90% of women with cervical carcinoma show evidence of HPV infections of the cervix. Accordingly, one possible alternative to conventional histopathological examination of cervical smears is to examine samples for evidence of HPV infection. For example, there have been numerous proposals to screen for cervical carcinoma by performing DNA hybridisation assays on samples, using nucleic acid probes specific for HPV sequences. Such hybridisation assays are generally favoured by those skilled in the art, because of the ready availability of suitable reagents and because of their high specificity.
Thus, for example in Fields Virology (Fields et al, [Eds.] Virology Vol. 2, p2099, 3rd Edn. (1996) Raven Press, New York), an authoritative virology text book, it is stated that xe2x80x9cDiagnosis of an HPV type in a tissue requires nucleic acid hybridization studiesxe2x80x9d.
In contrast, screening for cervical carcinoma by detection of expression of HPV polypeptides has generally been disregarded, being considered unsuitable for a number of reasons, primarily because of the difficulty in obtaining suitable reagents and, more significantly, many HPVs produce very little virus protein in mucosal infections, making detection difficult, uncertain and unreliable. Thus, in Fields Virology (cited above) it is stated that xe2x80x9cimmunologic detection of viral capsid antigensxe2x80x9d is xe2x80x9cof limited valuexe2x80x9d. The possibility of immunologic detection of other viral antigens is not even considered. If one were to develop a screening method based on detection of expression of viral proteins, the most likely choice of target would be those proteins which are best-characterised, such as L1 or L2. The function of E4 protein is at present unknown. Its expression pattern in cervical lesions has not been determined conclusively in the prior art so the molecule has not been an obvious choice for selection as a target for detecting HPV infection.
In accordance with the present invention, it has now been demonstrated that HPV infection can be detected in a sample taken from a patient by using molecules which bind specifically to E4 protein of HPVs. In particular, the invention provides a method of screening samples for pre-cancerous cervical lesions, using molecules which bind specifically to HPV E4 protein.
The present studies have clearly demonstrated HPV16 E4 protein to be cytoplasmic, and to be produced in cells supporting vegetative viral DNA replication.
In a first aspect the invention provides a method of detecting a papilloma virus infection in an organism, the method comprising the steps of: obtaining a sample of the organism""s cells from the site of potential papilloma virus infection; contacting the cells with a molecule that binds specifically to papilloma virus E4 protein; and monitoring said binding.
In particular, the invention provides a method of screening for pre-cancerous cervical lesions, comprising the steps of: obtaining a sample of cervical cells from a subject; contacting the cells with a molecule that binds specifically to HPV E4 protein; and monitoring said binding.
Moreover, the invention provides a method of determining the type(s) of HPV infection in a patient, the method comprising the steps of: obtaining a sample of the patient""s cells from the site of HPV infection; contacting the cells with a molecule that binds specifically to a subset of HPV E4 proteins; and monitoring said binding.
In a further aspect the invention provides an antibody molecule, or an antigen-binding variant thereof, which binds specifically to HPV E4 protein in the region of amino acid residues RPIPKPSPWAPKKHRRLSSDQDSQTP (SEQ ID NO:4) of HPV16 E4 protein, or the corresponding hydrophilic, acid/base-rich region of other HPV E4 proteins.
The invention moreover concerns the use of molecules capable of binding to E4 to target antiviral agents capable of destroying papilloma viruses and/or cells infected by papilloma viruses. Such molecules may be antibodies or peptides as described above and exemplified herein, optionally conjugated to anticancer or antiviral agents.